MICR 270 Mod 5 - Vaccines & Translational Immunology

Enzyme-Linked Immunoabsorbent Assay (ELISA) (how does it work?)

• ELISA is a fundamental tool of clinical immunology based on the principle of antigen-antibody interaction

1. the bottom of the wells are coated with an antigen that is specifically recognized by the antibody you wish to measure (primary antibody)

W - the wells are washed to remove any excess antigen not attached to the bottom of the well

2. the sample containing the antibody to be measured (ie. a patient’s serum) is added to the well. The primary antibodies, if present, will bind to the antigens attached to the bottom of the well

W - the wells are washed again to remove excess primary antibody not attached to the bound antigen as well as any other sample components that might interfere with subsequent steps

3. an enzyme-conjugated secondary antibody (a secondary antibody that specifically binds to the primary antibody. In this example, the secondary antibody used has an enzyme attached to it) is added to the well. This secondary antibody will bind to the Fc portion of the primary antibodies already present in the well. The secondary antibody used specifically recognizes antibodies from a specific animal (ie. anti-human, anti-rat, anti-rabit, etc)

W - the wells are washed to remove any excess secondary antibody no attached to the primary antibody

4. the substrate of the enzyme attached to the secondary antibody is added to the well. the reaction of the substrate (a chromogen, can be readily converted into dye) and the enzyme produces a coloured product that can be measured by absorbance

What does ELISA measure?

• an ELISA will measure a coloured reaction product by absorbance with the help of a machine called a spectrophotometric plate reader

• the data measured correlates with the presence of an antibody or an antigen

• this information can be used, for example, to detect the presence of a viral disease

Indirect ELSA

• detects or quantifies antibodies

  • ie. to determine the presence of serum antibodies against HIV

How does flow cytometry work?

• flow cytometry is a method of detecting and quantifying different cell types in a mixed cell suspension

• a narrow stream of cells in single file is passed through a laser light source

• the way laser light is scattered is unique to each cell type; this can be detected and analyzed'

• measuring FSC allows for the discrimination of cells by size

• FSC intensity is proportional to the diameter of the cell

• SSC provides information about the internal complexity (ie. granularity) of a cell

• for example, granules and the nucleus increase the side scatter of the laser in a flow cytometer

• when measured in conjunction, FSC and SSC allow for the detection of specific cells and cellular components within a heterogeneous population

• flow cytometry can also be used to determine the proportion of cells expressing a particular antigen

• in this case, cells are labelled with a specific antibody

• the antibody is coupled with a fluorescent marker. this marker can be excited by a light of a specific wavelength

• in turn, the fluorescent marker emits a light with a characteristic different wavelength

• only cells expressing this antibody in question will emit light of this specific wavelength

what does flow cytometry measure?

• flow cytometry is used to measure physical properties of a cell

• it can also be used to detect specific antigens on or inside a cell

• as each cell in a mixed suspension is assessed as an individual detection event, the total number of cells in the suspension, the number of cells of a particular type in the suspension, and the overall composition of the suspension can be readily determined

• flow cytometers are used to determine complete blood counts

clinical application of flow cytometry

• flow cytometry can be used to diagnose cancer in a variety of ways

• the main diagnostic tests focus on detecting DNA aneuploidy (abnormal number of chromosomes), analyzing cell cycles, and the immunophenotypical characterization

how do monoclonal antibodies work?

• monoclonal antibodies are antibodies that are produced by a single clone of a B cell that are specific for a single epitope (the portion of the antigen that is recognized and bound by an antibody)

• monoclonal antibodies are produced in the lab by hybridomas, immortal cells that produce unlimited quantities of one identical antibody

• hybridomas are the result of fusion between a plasma cell and a cancerous (or myeloma) cell

• hybridomas share properties of both plasma cells and myeloma cells

Myeloma cell: immortal growth, divides indefinitely

Plasma Cell: produces specific antibodies against one antigen

Hybridoma cell: a perpetual source of antibodies against one antigen

clinical applications of monoclonal antibodies

Immunotoxins

• immunotoxins consist of a tumour-specific monoclonal antibody attached to a deadly toxin

• this technique is still under investigation, but a long-term objective is to use immunotoxins to target and eliminate tumour cells and treat cancer

Radiolabelled Antibodies

• monoclonal antibodies tagged with a radioactive isotope can be used to diagnose tumours earlier that other methods

• radiolabelled antibodies can bind to antigens on a tumour thereby allowing the precise location of the tumour within the body to be visualized

Live-Attenuated Vaccine

Characteristics

• contains a modified strain of the disease causing agent which has lost its pathogenic ability, but retains its capacity to replicate within the host

Advantages

• provides a prolonged exposure to the disease causing agent, and its suitable to generate cell-mediated immunity

Disadvantages

• potential to revert to a virulent form

• requires specific storage and transport conditions (ie. refrigeration)

Examples

• smallpox vaccine

• oral poliovirus vaccine

• measles vaccine

Killed-Inactivated Vaccine

Characteristics

• contains a strain of the disease causing agent that has been inactivated by heat, chemicals, or radiation

• has the ability to generate an immune response, but is unable to replicate

Advantages

• safer option as it cannot mutate back into virulent form

• easy to store and transport

Disadvantages

• generally requires multiple booster doses to maintain immunity

• generally must be administered by injection

Examples

• rabies vaccine

• influenza vaccine

Toxoid Vaccine

characteristics

• contains an inactivated toxin which is a product from the pathogen that is causing the disease

Advantages

• safe as it is not a living organism that can divide, spread, and/or revert

• stable as they are less susceptible to changes in temperature, humidity, and light

Disadvantages

• may require several doses and usually need an adjuvant (a substance that enhances the body’s immune response to an antigen)

Examples

• tetanus vaccine

• diphtheria vaccine

Subunit Vaccine

Characteristics

• contains only a small part or fragment of the disease causing agent

Advantages

• the safest type of vaccine - can be used on everyone including immunocompromised, pregnant, or elderly populations

Disadvantages

• rarely successful at inducing long lasting immunity, which means it will require multiple booster doses to maintain immunity and might even need to be conjugated to a carrier

  • a carrier is a stronger antigen that the desired target antigen. By covalently attaching a strong antigen to a poor antigen, the overall immunological response is strengthened, and hopefully the immunological response to the poor antigen is also improved

Examples

• Hepatitis B vaccine

mRNA Vaccines

• mRNA vaccines are the most recent vaccine type that have changed the field of vaccinology

• they are known for their use against SARS-CoV-2, the COVID virus, and are used in several formulations to fight the virus (ie. bivalent vaccine, boosters)

• formulations of mRNA vaccines are also being investigated for other infectious diseases (ie. HIV, influenza), and non infectious clinical conditions (ie. pancreatic cancer, heart tissue regeneration)

• the principle of the assay relies on the use of mRNA to produce viral proteins and recruit immune cells to respond to the antigenic target

• the proteins are then displayed on the surface of an antigen presenting cell to induce B cells and T cell immunity

mRNA vaccine mechanism: vaccine production

• mRNA is made in the lab from a DNA template of the virus

• the mRNA encodes an antigen of the virus

  • for the COVID-19 vaccine, the mRNA encodes the spike protein on the surface of the virus

• the mRNA is incorporated into a formulation that can be administered as a vaccine

mRNA vaccine mechanism: host cell

• once inside the body, the mRNA enters the host cell and uses host cell machinery to produce the spike protein

mRNA vaccine mechanism: APC

• the newly formed spike protein exits the cell and is recognized by an APC

• the APC internalizes the spike protein and processes it into a peptide (or antigen)

• the APC then displays the antigen on the surface of the cell via the major histocompatibility complex

mRNA vaccine mechanism: immune response

• the antigen is recognized by a helper T cell, which initiates an immune response

• B cells produce antibodies that stop the virus from infecting cells

• T cells destroy cells infected with the virus

antiviral medications against COVID-19

Polymerase Inhibitor

• polymerase is an enzyme that plays a central role in viral replication and transcription

• Molnupiravir is a polymerase inhibitor used to treat COVID 19

• it increases the frequency of viral RNA mutations and impairs replication of the virus

Protease Inhibitor

• proteases cut proteins into smaller, more workable pieces

• protease inhibitors are often administered in combination

  • for example, nirmatreIvir stops protease from cutting viral proteins into functional pieces

  • Ritonavir protects nirmatreIvir from destruction by the body and allows it to keep working

• since these drugs disrupt the assembly of the virus, they can’t replicate and infect other cells

HPV VLP Vaccines

• VLPs (virus like particles) are composed of the structural proteins of HPV, which can self assemble into particles that resemble the natural virus both structurally and immunologically

• because they do not contain viral DNA, VLPs are not infectious

Ebola Vaccines

• Ebola virus (EBOV) and Marburg virus (MARV) produce transmembrane glycoproteins thought to play a role in the virulence of these viruses

• researchers have developed a potentially viable vaccine using glycoproteins from EBOV and putting them into a live attenuated recombinant vesicular stomatitis virus (VSV), that expresses the transmembrane glycoproteins of EBOV and MARV

Genital Herpes Vaccine

• a subunit vaccine was developed which was composed of a prominent structural protein of HSV-2

• this vaccine showed promise in early clinical trials, with reported efficacies of >70% in women who were seronegative for both HSV-1 and HSV-2 at the beginning of the trial

• a subsequent larger clinical trial however showed much lower efficacy (20%) and no protection from infection by HSV-2

phases of vaccine development: lab studies

• the first step in vaccine development is to identify the infectious agent causing the disease and select a strain (or subtype) which is relevant to the target population and will be used to produce the vaccine

• this stage is largely dependent on research carried out in the lab using assays, which may involve exhaustive screening to identify a suitable antigen and creation of a vaccine concept

• this stage also involves developing and testing the manufacturing process of the vaccine according to good manufacturing process standards

phases of vaccine development: preclinical studies

• preclinical studies involve research carried out in animal models to evaluate the pharmacological aspects of the product

• this is a stage of research that occurs before the clinical trials can begin

• at this point, the researchers will carry out challenge studies to demonstrate the immunogenicity of the vaccine in animal models

• immunogenicity means that the vaccine has the ability to induce an immune response which will prevent the development of the disease in case of subsequent infection with the pathogen

• another part of this stage is to carry out safety studies to evaluate the possible toxicity of the vaccine which would prevent its use in humans

phases of vaccine development: clinical phase I

• due to rigorous regulatory requirements, only a very small percentage of vaccines progress to licensing, making the costs of vaccine research and development extremely high

• Phase I clinical trials for vaccines involve small scale trials in humans (10-<100 people) to assess vaccine safety by evaluating local and systemic reactions after administration

• it also provides preliminary data on the immunogenicity of the vaccine and the immune response it evokes

phases of vaccine development: clinical phase II

• phase II trials occur at a bigger scale (50-500 people) to collect data on safety, side effects, and the efficacy of the vaccine

• this stage also evaluates the dosage requirements of the vaccine

  • clinical trial evaluators monitor the effects of increasing vaccine dosage and conduct challenge tests to define the optimal dose and vaccination schedule (ie. need for boosters) in target populations

phases of vaccine development: clinical phase III

• phase III trials involve multiple geographic sites with many hundreds of subjects (300-30,000 people) to evaluate efficacy under natural disease conditions

• in this step, researchers are required to demonstrate the efficacy in target populations and complete a safety assessment for the vaccine

• if the vaccine is successful in retaining safety and efficacy over a defined period of time, the manufacturer will then apply to the regulatory authorities for a license to market the product for human use

phases of vaccine development: health canada approval

• health canada is the regulatory authority in canada responsible for ensuring the quality, safety, and efficacy of all biological drugs, including vaccines for human use

• vaccine regulation is necessary as they are usually given to very large numbers of healthy individuals

• vaccines candidates must be submitted to health canada to be considered for approval with sufficient scientific and clinical evidence to show that it is safe, efficacious, and of suitable quality

• scientific evidence includes results form human clinical trials

• vaccines cannot be used clinically without approval from health canada

challenges with vaccine development

• high cost of the development of vaccines sometimes leads to premature abandonment of clinical research

• live attenuated vaccines, the most effective at producing long term immunological memory, need to be stored and transported in specific conditions

• the efficacy of a vaccine is directly related to specific antigens against which the immune response has developed

  • if the disease causing agents evolve by changing or losing their major antigenic determinants, the vaccine could lose its efficacy

• even if a vaccine has been approved, that doesn’t mean the work is done.

  • new strategies are developed to create better vaccines, for example, by using recombinant vector or improving vaccine adjuvant

vaccine development challenges: influenza virus

Neuroaminidase Antigen (NA)

• a surface protein that removes sialic acid from cell surfaces and enables new viral copies to infect and spread to other cells

• researchers have identified 11 NA subtypes, each with sequence variabilities in their receptor binding sites

Haemagglutinin Antigen (HA)

• a surface protein that recognizes and binds to sialic acid on cell surface glycoproteins

• these HA cell surface interactions lead to endocytosis of the virus, and the HAs are activated to fuse the endosome and viral membrane

• researchers have identified 18 HA subtypes, each with sequence variabilities in their receptor binding sites

herd immunity

• herd immunity can be measured by calculating what is called the basic reproduction number, R0

• R0 is defined as the average number of secondary cases of an infectious disease arising from a typical case in a totally susceptible population

• R0 determines the herd immunity threshold and therefore the immunization coverage required to achieve elimination of an infectious disease

• as R0 increases, higher immunization coverage is required to achieve herd immunity

introduction to cancer

cancer cells vs normal cells

• cancer cells behave differently than normal cells of the body

• they do not need specific growth factors to divide, which normal cells do

• they also don’t respond to the signals that cause normal cells to stop dividing

• in the field of immunology, cancer cells are viewed as self cells that have been altered to escape the normal growth-regulating mechanisms

• these changes are the results of alterations in DNA which induce cell transformation to malignant cells

Tumour

• cancer cells continue to divide and grow, ultimately forming a tumour

• a tumour is an abnormal mass in tissue

Cancer Immunotherapy

• cancer treatment approaches that are either based on secreted or cellular components of the immune system are broadly defined as immunotherapies

• these therapies are primarily aimed at enhancing host anti tumour immune responses and have recently joined the pillars of cancer management

tumours

benign tumour

• not cancerous

• unable to grow indefinitely or invade surrounding tissues

malignant tumour

• cancerous

• ability to metastasize

• continuous growth

the resilience of cancer cells

• cancer cells show resilience in environments that a normal cell might not survive

HeLa Cells

• HeLa cells are a line of cancerous cells which were cultured from Henrietta Lacks in 1951

  • these cells were the first line of human cell culture to survive outside the human body

• the resilience of HeLa cells allowed for their immortality, thus the cell cultures are an important tool for scientific discovery to this day

HeLa Cells: research breakthroughs

Recombinant Protein Production

• although bacterial cells can be utilized for protein production, they lack the required mechanics to produce more complex proteins. The HeLa cell line allowed researchers to overcome this challenge

HPV Vaccine

• researchers found that HeLa cells were infected with HPV-18 virus, and decades of characterizing HPV-18 led to the development of HPV vaccines

Understanding Virology

• experimental viral infection on HeLa cells allowed researchers to characterize how specific viruses can evade the immune system, ie. CD4 T cell receptor utilization by HIV

Toxicity Testing

• originally, hepatocytes were used to test toxicity, but proved too unstable for sustained use. the resilient HeLa cells are now used to test cytotoxicity of drugs, an important practice in drug development and discovery

Monoclonal Antibody (mAB) production

• mABs, which can be produced using hybridoma crosses of HeLa and other animal cells, have many applications such as medical diagnoses and cancer therapy

Polio Vaccine

• this vaccine was developed in the 1950s, but HeLa cells were the only human cells that could be used to test the vaccine

Genome Sequencing

• HeLa cells fused with mouse cells became the first hybrid cells, the fusion of two cell types, in research. This breakthrough aided in the emergence of the human genome project

Telomerase Activity

• in 1996, scientist Gregg Morin isolated telomerase from HeLa cells which were previously only found in animal embryos. This supported his hypothesis that both embryos and cancer cells utilize telomerase to rapidly divide, giving researchers insight on the importance of telomerase in human embryology

the cancer-immunity cycle stage 1: release of cancer cell antigens

• cancer cell death

• antigens are released by mutated cancer cells, indicating that they are not healthy cells

• the immune system is able to recognize these antigens

the cancer-immunity cycle stage 2: cancer antigen presentation

• dendritic cells/APCs

• the cells of the immune system capture the released antigens and travel to the lymph nodes where they find T cells

the cancer-immunity cycle stage 3: priming and activation

• APCs & T cells

• T cells are activated by the antigens and the immune response against the cancer is initiated

the cancer-immunity cycle stage 4: trafficking of T cells to tumours (CTLs)

• the activated T cells move through the blood vessels to the site of the tumour

the cancer-immunity cycle stage 5: infiltration of T cells into tumours

• CTLs, endothelial cells

• once the T cells reach the cancerous cells, they invade the tumour and attack it

the cancer-immunity cycle stage 6: recognition of cancer cells by T cells

• CTLs, cancer cells

• T cells recognize cancer cells because of the antigens they had previously released

the cancer-immunity cycle stage 7: killing of cancer cells

• immune & cancer cells

• T cells initiate a pathway that results in cancer cell death

tumour immunosurveillance vs immunoediting

Immunosurveillance

• theory of immunosurveillance states that tumour cells are identified and kept under control by the immune systems of healthy individuals

• however, cancer cells sometimes evade recognition by the immune system, or the magnitude of the antigen-tumour immune response is not sufficient to kill all of the cancer cells

• recent research has even shown that often the immune environment around the cancer cells can promote tumour progression

Immunoediting

• immunoediting is. adynamic process which describes the connection between the tumour cells and the immune system in the context of immunosurveillance and tumour progression

• during cancer evolution, there is ongoing cross talk between the cancer cells and the immune cells

• the immune system destroys the growing cancer cells, but this promotes tumour growth in the cells which have the ability to evade immunosurveillance

• cancer immunoediting is constituted by 3 phases:

  • elimination

  • equilibrium

  • escape

cancer immunoediting: elimination

• when a tumour cell arises in a tissue, the immune system can quickly act to remove it

• a variety of immune cells, including NK cells, cytotoxic T cells, and helper T cells, can recognize the altered cell and work to eliminate it

cancer immunoediting: equilibrium

• if the tumour cells are not eliminated, they can enter a state of equilibrium where the cell proliferation is matched by cell killing by the immune system

• this phase can last for a short time or many years

cancer immunoediting: escape

• tumour cells are no longer recognized by the immune system, so avoid elimination

• these cells are able to grown uncontrolled and eventually proliferate to form a tumour

cancer evasion of the immune response

reduced MHC expression

• tumour cells display low levels of MHC class I molecules on their cell surface

• as cytotoxic T lymphocytes (CTLs) recognize antigens in the context of MHC class I molecules, an absence of these molecules will inhibit recognition of tumour cells

Poor Costimulatory Molecules

• T cells require both expression of MHC and costimulatory molecules to become activated

• tumours lack these costimulatory molecules, which contribute to their poor immunogenicity

• T cells will only be partially activated

cancer immunotherapy

• immunotherapy is able to attack cancerous cells throughout all organs in the body

• immunotherapy allows the immune system to specifically target and eliminate cancer cells without damaging healthy cells, resulting in fewer side effects that traditional cancer treatments

• immunotherapy takes advantage of immunological memory, allowing for the possibility of long term protection

• immunotherapy can be applied to almost all types of cancer

tumour infiltrating lymphocytes (TILs)

• although T cells are shown to be the most effective lymphocyte population in killing cancer cells, B cell lymphocytes are also important. these are broadly termed tumour infiltrating lymphocytes

• the type, density, and location of TILs has been suggested as. a prognostic biomarker in some cancers

• TILs leave the bloodstream and migrate to infiltrate the tumour under influence of various chemotactic gradients of specific types of chemokines

• TILs can be a mix of T and B cells. NK cells, dendritic cells, and macrophages also can be of prognostic relevance

• it is important to note that the number of TILs is not always reflective of their activity and prognostic significance

• some of the dynamic phenotypic markers expressed on these cells reflect their activation status and therefore must accompany any interpretation on their roles as biomarkers for diagnosis, such as in the case of breast cancer

Prognostic biomarker: biological characteristics that are objectively measured and evaluated to predict the course of a disease or response to a therapeutic intervention among patients with the same characteristic

Biomarkers: a measurable substance in an organism, the presence of which is indicative of some phenomenon such as disease, infection, or environmental exposure

immunological classification of tumours: T cell inflamed “hot” tumours

• hot tumours show compartively higher immune activity compared to cold tumours

• characteristics:

  • high numbers of CD8+ TILs

  • high levels of interferon IFN genes

  • usually respond well to treatment (chemo or immunotherapy)

immunological classification of tumours: T cell non inflamed “cold” tumours

• cold tumours show lower immune activity compared to hot tumours

• characteristics:

  • low numbers of CD8+ TILs

  • low levels of interferon (IFN) genes

  • usually inferior response to treatment (immunotherapy or chemo)

treatment of cold and hot tumours

• because of this immunological difference between hot and cold tumours, conceptually, one could convert cold tumours. to hot by stimulating the tumour interferon activity

• this knowledge has led to the exploration of anti tumour immunity towards development of newer treatments

the immunoscore

• combining the knowledge of TILs and the immunological classification of hot and cold tumours has led scientists to develop a new and reliable prognostic biomarker for cancer diagnosis, such as in the case of colon cancer

• an immunoscore measures the density/numbers of T cells in the centre (CT) and in the periphery (IM) of the tumour by immunochemistry

• this can help stratify patients as having high risk or low risk cancers, and aid in developing treatment plans

Workflow for determining an immunoscore:

1. separating the tumour in the central (CT) and peripheral (IM) regions

2. staining for T cells and conducting digital pathology

3. assigning a score to the tumour to relate it with an associated diagnosis or risk attribution

immunotherapy approaches based on tumour immune microenvironment

• the immune system can be activated through a variety of ways to combat tumours

• there are many other therapies under development that exploit the components of the immune system to fight cancer, and the field is constantly innovating and developing solutions to this complex health issue